Identifying enzymes involved in Clostridium thermocellum ferredoxin metabolism

Engineering microbes for biofuels production requires understanding of enzymes involved in electron metabolism. Anaerobic metabolism  Here, we identify enzymes important for ethanol formation in Clostridium thermocellum, focusing on enzymes involved in ferredoxin metabolism, including hydrogenases, the Rnf complex, NfnAB, and pyruvate ferredoxin oxidoreductase. Using genetic deletions, we target each of these enzymes, alone and in combination, and determine their relative importance to fermentation products. We show that loss of the Rnf complex causes a 40% reduction in the amount of ethanol formed versus the parent strain. Loss of NfnAB has a minimal impact on fermentation products formed, unless other mechanisms of ferredoxin reoxidation are lost or inhibited. We target two annotated pyruvate ferredoxin oxidoreductases and show that one of these is primarily responsible for pyruvate metabolism.